The Question: What are the epigenetic mechanisms that assist a pre-malignant cell in acquisition of the cancer characteristics and thus promote cancer progression? The emerging importance of epigenetic gene regulation in cancer progression necessitates not only our understanding of which genes are potential targets, but what mechanisms are employed in targeting those genes. Understanding the mechanisms that promote evolution of the normal genome and epigenome into the malignancy of cancer are central to our advancement in understanding cancer. Thus, we propose to investigate canalization as a mechanism of epigenetic gene regulation in a mammalian system of cancer progression, which we initially discovered in our studies of Drosophila. The results of our investigations in the Drosophila model system indicate a very important gene in cancer progression, heat shock protein 90 (Hsp90), is well connected to epigenetic gene regulation and signal transduction cascades. Hsp90 is over-expressed in multiple cancers with poor prognosis, including acute myelogenous leukemia (AML). Current theory is that Hsp90 has a protective function for pre-malignant cells that promotes cancer progression. The Hypothesis: This proposal is designed to test the hypothesis that inhibition of HSP90 results in increased cell plasticity associated with loss of canalization mediated by an epigenetic mechanism that can promote early progression in acute myelogenous leukemia. Research Prospectus: The approach is to test the hypothesis in vitro and then test the effects of loss of canalization on cancer progression in vivo. We will exploit the connection between epigenetics and cellular differentiation in studies of differentiation from stem cells to progenitor, monocyte, and macrophage cells. The major tools of investigation will be flow cytometry, cell proliferation assays, and measures of apoptotic rates using the EML selection point model we have developed. In specific aim 1, Modulation of Hsp90 levels will be performed in this system followed by analyses of the ability of hematopoietic cells to adapt in a restrictive cell culture medium. We will investigate the granulocyte-macrophage colony stimulating factor signal transduction cascades to determine if canalization is affecting its gene targets. In specific aim 2 we will test the role of epigenetics and identify genes involved in mediating the process of canalization using our model. In specific aim 3, we will test our assertation that loss of canalization can promote cancer in an in vivo model of AML. Benefit: This highly innovative proposal can yield a paradigmatic influence on how epigenetic regulation drives oncogenesis. Our increased understanding of the role of HSP90 in cancer progression will facilitate the development of new combinatorial therapeutic strategies for most cancers.